1. Technical Field
The present invention relates to methods and compositions for systemic introduction of exogenous genetic material into mammalian, particularly human, cells in vivo.
2. Background
An ever-expanding array of genes for which abnormal expression is associated with life-threatening human diseases is being cloned and identified. The ability to express such cloned genes in humans will ultimately permit the prevention and/or cure of many important human diseases, diseases which now either are treated poorly or are untreatable by currently available therapies. As an example, in vivo expression of cholesterol-regulating genes, genes which selectively block the replication of HIV, or of tumor-suppressing genes in human patients should dramatically improve treatment of heart disease, HIV, and cancer, respectively. However, currently available gene delivery strategies have been unable to produce either a high level of or generalized transgene expression in vivo in a wide variety of tissues after systemic administration to a mammalian host. This inability has precluded the development of effective gene therapy for most human diseases.
Approaches to gene therapy include both different goals and different means of achieving those goals. The goals generally include gene replacement, gene correction and gene augmentation. In gene replacement, a mutant gene sequence is specifically removed from the genome and replaced with a normal, functional gene. In gene correction, a mutant gene sequence is corrected without any additional changes in the target genome. In gene augmentation, the expression of mutant genes in defective cells is modified by introducing foreign normal genetic sequences.
The means to reach the above goals used by others, have included xe2x80x9cex vivoxe2x80x9d transfection of a target cell followed by introduction of the transformed cells into a suitable organ in the host mammal. Ex vivo techniques include transfection of cells in vitro with either naked DNA or DNA liposome conjugates, followed by introduction into a host organ (xe2x80x9cex vivoxe2x80x9d gene therapy). The criteria for a suitable target organ or tissue include that the target organ or tissue is easily accessible, that it can be manipulated in vitro, that it is susceptible to genetic modification methods and ideally, it should contain either non-replicating cells or cycling stem cells to perpetuate a genetic correction. Further, it should be possible to reimplant the genetically modified cells into the organism in a functional and stable form. Exemplary of a target organ which meets these criteria is the mammalian bone marrow. A further criterion for ex vivo gene therapy, if for example a retroviral vector is used, is that the cells be pre-mitotic; post-mitotic cells are refractory to infection with retroviral vectors. Although this has not been reported, in some instances it may be possible to transfect cells from other than the target organ or tissue using ex vivo gene therapy if the corrective gene product can be secreted and exert the desired effect on/in the target cell following circulation in blood or other body fluids.
There are several drawbacks to ex vivo therapy; for example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not removed first from the body. The above methods generally involve integration of new genetic material into the cell genome and thus constitute permanent changes.
Liposomes have been used effectively, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcription factors and other cellular effectors into a variety of cultured cell lines and animals. The agent to be introduced is typically entrapped within the liposome, or lipid vesicle, or the agent may be bound to the outside of the vesicle. Successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed. Several strategies have been devised to increase the effectiveness of liposome-mediated drug delivery by targeting liposomes to specific tissues and specific cell types. However, while the basic methodology for using liposome-mediated vectors is well developed, the technique has not been perfected for liposome-based transfection vectors for in vivo gene therapy.
In vivo expression of transgenes as reported by others has been restricted to injection of transgenes directly into a specific tissue, such as direct intratracheal, intramuscular or intraarterial injection of naked DNA or of DNA-cationic liposome complexes, or to ex vivo transfection of host cells, with subsequent reinfusion. The expression is low and generally has been limited to one tissue, typically the tissue that was injected (for example muscle); liver or lung where iv injection has been used; or lung where intratracheal injection has been used, and less than 1% of all cells within these tissues were transfected. In some cases, transfection of cells has been obtained in tissues afferent to the site of intravenous administration.
Currently available gene delivery strategies consistently have failed to produce a high level and/or generalized transgene expression in vivo. It therefore would be of interest to develop compositions and delivery methods for in vivo gene therapy that provide for a high level of transcription of the transgene and/or expression in a variety of cell and tissue types for the in vivo treatment, prevention, or palliation of numerous human diseases. Also of interest, is the use of gene modulation as an alternate means of gene therapy. In gene modulation, expression of an already expressed gene is increased by introducing exogenous normal genetic sequences and decreased by introducing antisense genes or gene fragments, or by introducing vectors that can produce ribozymes that can cleave specific mRNAs. Gene modulation can also be achieved by the introduction of exogenous normal genetic sequences that code for proteins that modulate the extent of gene expression, or affect the processing, assembly or secretion of gene products.
Relevant Literature
A large number of publications relate to in vivo and ex vivo transfection of mammals. In some cases, only transcription of a transgene has been achieved, in others, the data appear to show only a low level of expression and/or expression in a limited number of tissues or cell types. The following are examples of the publications in this area.
A variety of approaches for introducing functional new genetic material into cells, both in vitro and in vivo have been attempted (Friedmann, (1989) Science, 244:1275-1281). These approaches include integration of the gene to be expressed into modified retroviruses (Friedmann, (1989) supra; Rosenberg, (1991) Cancer Research, 51(18), Suppl.: 5074S-5079S); integration into non-retrovirus vectors (Rosenfeld, et al., (1992) Cell, 68:143-155; Rosenfeld, et al., (1991) Science, 252:431-434); or delivery of a transgene linked to a heterologous promoter-enhancer element via liposomes (Friedmann, (1989), supra; Brigham, et al., (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al., (1990) Science, 249:1285-1288; Hazinski, et al., (1991) Am. J. Resp. Cell Molec. Biol., 4:206-209; and Wang and Huang, (1987) Proc. Natl. Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific, cation-based transport systems (Wu and Wu, (1988) J. Biol. Chem., 263:14621-14624) or the use of naked DNA expression vectors (Nabel et al., (1990), supra); Wolff et al., (1990) Science, 247:1465-1468). Direct injection of transgenes into tissue produces only localized expression (Rosenfeld, (1992) supra); Rosenfeld et al., (1991) supra; Brigham et al., (1989) supra; Nabel, (1990) supra; and Hazinski et al., (1991) supra). The Brigham et al. group (Am. J. Med. Sci., (1989) 298:278-281 and Clinical Research, (1991) 39 (abstract)) have reported in vivo transfection only of lungs of mice following either intravenous or intratracheal administration of a DNA liposome complex. An example of a review article of human gene therapy procedures is: Anderson, (1992) Science 256:808-813.
PCT/US90/01515 (Felgner et al.) is directed to methods for delivering a gene coding for a pharmaceutical or immunogenic polypeptide to the interior of a cell of a vertebrate in vivo. Expression of the transgenes is limited to the tissue of injection. PCT/US90/05993 (Brigham) is directed to a method for obtaining expression of a transgene in mammalian lung cells following either iv or intratracheal injection of an expression construct. PCT 89/02469 and PCT 90/06997 are directed to ex vivo gene therapy, which is limited to expressing a transgene in cells that can be taken out of the body such as lymphocytes. PCT 89/12109 is likewise directed to ex vivo gene therapy. PCT 90/12878 is directed to an enhancer which provides a high level of expression both in transformed cell lines and in transgenic mice using ex vivo transfection. PCT/US92/08806 is directed to particle-mediated transformation of mammalian unattached cells. EP application 91301819.8 is directed to the use of recombinant techniques to produce cystic fibrosis transmembrane conductance regulator (CFTR).
Methods and compositions are provided for introduction of a transgene into a plurality of mammalian tissues in vivo. The method includes the step of incorporating a transfection cassette comprising a nucleotide sequence of interest into a largely non-integrating plasmid and introducing the plasmid into a mammalian host, other than by directly introducing it into a specific tissue. The transfection cassette comprises as operably joined components, a transcriptional and initiation regulatory region, a nucleic acid sequence of interest, and a transcriptional termination regulatory region, wherein said regulatory regions are functional in the cells of a mammalian host, and wherein said nucleic acid sequence of interest is free of introns. Optionally, the transcriptional cassette is complexed with a cationic lipid carrier. The method finds use to modulate the phenotype of mammalian cells.